CN102597771A - High affinity adaptor molecules for redirecting antibody specifity - Google Patents

Abstract

Disclosed are methods for identifying high affinity adaptor molecules that bind to both a circulating antibody and a target molecule and redirect the specificity of the circulating antibody to the target molecule. Exemplary high affinity adaptor molecules are also provided.

Description

High affinity adaptor molecules for redirecting antibody specificity

RELATED APPLICATIONS

The present application claims priority from U.S. provisional application serial No. 61/248,778 filed on 5/10/2009 and U.S. provisional application serial No. 61/257,351 filed on 2/11/2009. The entire contents of the above application are incorporated herein by reference.

Background

The concept of redirecting the immune system to attack new targets has long been of interest to scientists as an attractive strategy for targeted immunotherapy. By redirecting natural circulating human antibodies to attack desired targets in disease regions such as cancer, autoimmune diseases, and infectious diseases, one can avoid the need for special immunity. Although this strategy has shown early signs of success, most studies have not exceeded the level of in vitro validation. For example, if antibodies are used that are already present in the general population, this strategy would be particularly valuable and could be prepared for oral administration. Accordingly, there is a need in the art for improved methods of redirecting antibody specificity.

Disclosure of Invention

The present invention provides isolated adaptor molecules, in particular bi-specific adaptor peptides, that bind antibodies and desired target molecules with high binding affinity and selectivity. Due to their high binding affinity and selectivity, the adaptor molecules of the present invention are able to efficiently redirect circulating antibodies to target molecules that are not normally bound by antibody molecules. Furthermore, the adaptor molecules disclosed herein provide one or more of the following advantages over traditional antibody-mediated therapies: 1) enabling the simultaneous recruitment of multiple antibody-like effector functions; 2) can be produced quickly using the method of the invention; 3) has low commodity cost; and 4) does not cause an IgE-mediated hypersensitivity reaction when administered to a subject.

The adapter molecules typically comprise one or more targeting moieties linked to one or more ligand moieties. In certain embodiments, the ligand moiety comprises one or more Gal antigens (e.g., Gal- α -1-3-Gal) or mimetics thereof. In one embodiment, the targeting moiety comprises a peptide, such as a VEGF or TNF α -binding peptide. Exemplary VEGF-binding peptides have one or more amino acid sequences as set forth in SEQ ID NO.1, 2, 3, and/or 4. In another preferred embodiment, the peptide (e.g., a peptide sequence as set forth in SEQ ID No.1, 2, 3, and/or 4) is linked to a ligand moiety comprising one or more Gal antigens (e.g., Gal- α -1-3-Gal disaccharide).

In other embodiments, the targeting moiety comprises one or more antibodies or antigen binding fragments thereof. Suitable antibodies include, but are not limited to, Abciximab (Abciximab), Adalimumab (Adalimumab), Alemtuzumab (Alemtuzumab), Basiliximab (Basiliximab), Bevacizumab (Bevacizumab), Cetuximab (Cetuximab), Cetuximab (Certolizumab pegol), Daclizumab (Daclizumab), Eculizumab (Eculizumab), efulizumab (Efalizumab), Gemtuzumab (Gemtuzumab), Ibritumomab (Ibritumomab tiuxetan), Infliximab (Infliximab), Muromonab (Muromonab) -CD3, Natalizumab (Natalizumab), Omalizumab (omab), Palivizumab (Palivizumab), rituzumab (Rituximab), Rituximab (Rituximab), or a fragment thereof. In a preferred embodiment, an antibody (e.g., one or more of the above-disclosed antibodies) or antigen-binding fragment thereof is linked to a ligand moiety comprising one or more Gal antigens (e.g., Gal- α -1-3-Gal). In another preferred embodiment, an antibody (e.g., one or more of the above-disclosed antibodies) or antigen-binding fragment thereof is linked to a ligand moiety comprising one Gal- α -1-3-Gal disaccharide. In another preferred embodiment, a ligand moiety comprising one or more Gal antigens (e.g., Gal- α -1-3-Gal) is linked to one or more variable regions of an antibody.

In other embodiments, the targeting moiety comprises an antibody-like molecule. Suitable antibody-like molecules include, but are not limited to, Adnectins, Affibodies, DARPins, antibodies, Avimers, and Versabodies, or antigen-binding fragments thereof. In a preferred embodiment, the antibody-like molecule is linked to a ligand moiety comprising one or more Gal antigens (e.g., Gal- α -1-3-Gal).

In other embodiments, the targeting moiety of the present invention comprises an extracellular portion of a cell surface receptor or fragment thereof. Suitable cell surface receptors include, but are not limited to, TNF family receptors (e.g., TNF α receptors, e.g., human TNF α receptors) and growth factor receptors of the tyrosine kinase family (e.g., p185HER 2). In a preferred embodiment, the extracellular portion of the cell surface receptor molecule is linked to a ligand moiety comprising one or more Gal antigens (e.g., Gal- α -1-3-Gal). In another preferred embodiment, the extracellular portion of the cell surface receptor molecule is linked to a ligand moiety comprising one Gal- α -1-3-Gal disaccharide.

In other embodiments, the targeting moiety comprises a ligand for a cell surface receptor. In a preferred embodiment, the ligand is linked to a ligand moiety comprising one or more Gal antigens (e.g., Gal- α -1-3-Gal). In another preferred embodiment, the ligand is linked to a ligand moiety comprising one Gal- α -1-3-Gal disaccharide.

In another aspect, the invention also provides methods for identifying isolated adaptor molecules, such methods comprising: providing a randomized library encoding a population of candidate targeting moieties; selecting from the display library targeting moieties that bind with high affinity and/or selectivity to the target molecule; ligating the targeting moiety and the ligand moiety through a linking moiety to form a candidate adaptor molecule; and evaluating the ability of the candidate adaptor molecule to redirect the specificity of the circulating antibody to the target molecule. Suitable screening methods for use in the methods of the invention include mRNA display, ribosome display, yeast display, phage display or screening of synthetic peptide libraries. In a preferred embodiment, an mRNA display library is used to select for targeting peptides. In another preferred embodiment, a phage display library is used to select for targeting peptides.

A further aspect of the invention provides a method of treating a disease (e.g., cancer, infectious disease, or autoimmune disease) in a subject, comprising administering to the subject an effective amount of an isolated adaptor molecule of the invention, thereby treating the disease. In a particular embodiment, the disease is at least one of macular degeneration, diabetic retinopathy, psoriasis, diabetes mellitus, cardiovascular ischemia, psoriasis, rheumatoid arthritis, and osteoarthritis.

Drawings

FIG. 1 is a schematic representation of an exemplary adaptor molecule of the present invention. The adaptor molecule is a bispecific peptide comprising a high affinity peptide targeting moiety directed against a target of interest and a ligand moiety comprising a glycopeptide epitope of an anti-gal antibody. By binding the target molecule and the naturally occurring anti-gal antibody, the adaptor molecule is able to redirect the effector functions of the antibody to act on the target molecule.

Figure 2 provides an overview of the various method steps performed during mRNA display.

Figure 3 depicts an exemplary peptide library that can be used in mRNA display for selection of high affinity adapter peptides.

Figure 4 depicts four exemplary high affinity VEGF targeting peptides of the invention.

FIG. 5 depicts exemplary methods and linkers for coupling a targeting moiety (e.g., a VEGF targeting moiety of the invention) to a ligand moiety.

FIG. 6 depicts HPLC and mass spectrometry analysis of the target moiety/ligand moiety coupling reaction.

Figure 7 depicts HPLC and mass spectrometry analysis of optimized target moiety/ligand moiety coupling reactions.

FIG. 8 depicts the results of an in vitro assay demonstrating the efficacy of the VEGF-binding adaptors of the present invention to redirect naturally occurring anti-gal antibodies to bind VEGF.

Detailed Description

The specification describes, inter alia, the identification and preparation of novel adaptor molecules that bind antibodies and target molecules with high binding affinity and selectivity. As used herein, the term "adaptor" refers to the ability of a peptide to facilitate a functional interaction between an antibody and a target molecule to which the antibody is not normally bound. For example, the adaptor molecules of the present invention are capable of binding an antibody and a molecule of interest such that the antibody and the target are in close proximity to each other. In some embodiments, the antibody is capable of promoting an antibody response to a target molecule. Exemplary antibody responses may include neutralization or opsonization of a target molecule (e.g., a soluble factor such as a cytokine or growth factor). Alternatively, the antibody may promote neutralization or opsonization of the virus when the target molecule is present on the surface of the virus. In other embodiments, the molecule of interest may be present on the surface of a cell (e.g., an infected cell or tumor cell) and recruitment of the antibody to the cell via an adaptor peptide facilitates induction of an antibody-mediated effector response (e.g., induction of the complement cascade or antibody-dependent cellular cytotoxicity (ADCC)). The adaptor molecules disclosed herein are particularly advantageous as they do not significantly activate basophils and therefore do not cause IgE hypersensitivity when administered to a subject.

Adapter molecules of the invention comprise at least three moieties: (a) a targeting moiety, (b) a ligand moiety, and (c) a linker moiety. The targeting moiety (a) is a moiety that binds with high affinity and/or selectivity to the target molecule. The ligand moiety (b) is a moiety to which the circulating antibody binds with high affinity and/or selectivity. The targeting moiety and the ligand moiety are operably linked by an intervening linker moiety (c). The linker moiety may be a covalent bond, a chemical linker or a peptide amino acid sequence, or any other moiety capable of linking the targeting moiety and the ligand moiety of the adapter peptide.

(a) Targeting moieties

Targeting moieties of the invention are selected for their ability to bind with high affinity and/or selectivity to the target molecule. In a particular embodimentThe targeting moiety specifically binds to the target molecule with a dissociation constant (KD) of 100 nanomolar or less (e.g., 10nM or less, 1nM or less, 100pM or less, 10pM or less, or 1pM or less). In other embodiments, the targeting molecule exhibits high selectivity. By "specifically binds" is meant that the moiety recognizes and interacts with the target molecule, but does not substantially recognize and interact with other molecules in the sample, such as a biological sample. In particular embodiments, the binding affinity for the targeting moiety of the target molecule is at least 1000-fold higher (e.g., 10-fold higher) than the binding affinity for the non-target molecule³10 times of⁴10 times of⁵10 times of⁶Multiple or 10 times⁷Multiple).

The targeting moiety may be selected for its ability to bind with high selectivity and/or affinity to virtually any target molecule. In particular embodiments, the targeting moiety binds to a pathogen-associated target molecule, including but not limited to a surface protein or antigen from a virus (e.g., HAV, HBV, or HCV, HIV, influenza), bacteria, yeast, parasite, or fungus. In other embodiments, the target molecule is a cell surface protein, including but not limited to a cell surface antigen or receptor from an infected host cell or tumor cell. Exemplary tumor-associated antigens include the growth factor receptor of the tyrosine kinase family, p185HER 2. In other embodiments, the target molecule is a hormone or growth factor. Exemplary hormones or growth factors include tumor necrosis factor alpha (TNF α) or Vascular Endothelial Growth Factor (VEGF). In other embodiments, the target molecule is an antibody (e.g., an autoantibody).

In a preferred embodiment, the targeting moiety of the present invention comprises a peptide moiety. The length of the peptide targeting moiety is desirably at least 3-200 amino acids, preferably at least 3-100 amino acids, more preferably 3-50 amino acids, and even more preferably 3-30 amino acids (e.g., 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids). Exemplary peptides for use as targeting moieties are described in WO/2010014830, the entire contents of which are incorporated herein by reference. In a preferred embodiment, the peptide is a VEGF-binding peptide or VEGF-binding portion thereof comprising or consisting of any one or more of the following amino acid sequences:

SEQ ID NO.1

H-Gly-Val-Gln-Glu-Asp-Val-Ser-Ser-Thr-Leu-Gly-Ser-Trp-Val-Leu-Leu-Pro-Phe-His-Arg-Gly-Thr-Arg-Leu-Ser-Val-Trp-Val-Thr

SEQ ID NO.2

H-Gly-Gly-Phe-Glu-Gly-Leu-Ser-Gln-Ala-Arg-Lys-Asp-Gln-Leu-Trp-Leu-Phe-Leu-Met-Gln-His-Ile-Arg-Ser-Tyr-Arg-Thr-Ile-Thr

SEQ ID NO.3

H-Gly-Val-Gly-Gly-Ser-Arg-Leu-Glu-Ala-Tyr-Lys-Lys-Asp-His-Arg-Val-Phe-Gln-Met-Ala-Trp-Leu-Gln-Tyr-Trp-Ser-Thr-Thr; and/or

SEQ ID NO.4

H-Gly-Ser-Gly-Ser-Gly-Asn-Ala-Leu-His-Trp-Val-Cys-Ala-Ser-Asn-Ile-Cys-Trp-Arg-Thr-Pro-Trp-Ala-Gly-Gln-Leu-Trp-Gly-Leu-Val-Arg-Leu-Thr

In other embodiments, the targeting moiety of the invention comprises an antibody, or binding fragment thereof (e.g., a CDR (e.g., CDRH3), variable domain (VH or VL), or Fab fragment). Any antibody or fragment thereof from any animal species is contemplated for use in the methods and compositions described herein. Suitable antibodies and antibody fragments include, but are not limited to, single chain antibodies (see, e.g., Bird et al (1988) Science 242: 423-426; and Huston et al (1988) Proc. Natl. Acad. Sci. U.S. A85: 5879-5883, each of which is incorporated herein by reference), domain antibodies (see, e.g., U.S. Pat. No. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245, each of which is incorporated herein by reference), Nanobodies (see, e.g., U.S. Pat. No. 6,765,087, each of which is incorporated herein by reference), and UniBodies (see, e.g., WO2007/059782, each of which is incorporated herein by reference). In some embodiments, the antibody is abciximab, adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, pegcetuximab, daclizumab, eculizumab, efletuzumab, gemtuzumab, ibritumomab tiuxetan, infliximab, moluzumab-CD 3, natalizumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tositumomab, trastuzumab, and/or golimumab, or an antigen-binding fragment thereof.

In other embodiments, the targeting moiety of the present invention comprises an antibody-like molecule. Suitable antibody-like molecules include, but are not limited to, Adnectins (see, e.g., WO 2009/083804, the entire contents of which are incorporated herein by reference), affibodies (see, e.g., U.S. patent No.5,831,012, the entire contents of which are incorporated herein by reference), DARPins (see, e.g., U.S. patent application publication No. 2004/0132028, the entire contents of which are incorporated herein by reference), antibodies (see, e.g., U.S. patent application publication No. 7,250,297, the entire contents of which are incorporated herein by reference), Avimers (see, e.g., U.S. patent application publication No. 200610286603, the entire contents of which are incorporated herein by reference), and Versabodies (see, e.g., U.S. patent application publication No. 2007/0191272, the entire contents of which are incorporated herein by reference).

In other embodiments, the targeting moieties of the invention comprise a ligand for a cell surface receptor, wherein the ligand is capable of recruiting an adaptor molecule to a cell expressing the cell surface receptor.

In other embodiments, the targeting moiety of the invention comprises an extracellular portion of a cell surface receptor or fragment thereof, wherein the cell surface receptor or fragment thereof is capable of recruiting an adaptor molecule to a cognate ligand of the cell surface receptor. Suitable cell surface receptors include, but are not limited to, TNF family receptors (e.g., TNF α receptors, e.g., human TNF α receptors) and growth factor receptors of the tyrosine kinase family (e.g., p185HER 2).

In some exemplary embodiments, the targeting moiety of the invention comprises an Fc fusion protein or immunoadhesin (e.g., a TNF receptor-Fc fusion, such as etanercept).

(b) Ligand moieties

The ligand portion of the invention comprises an antigenic domain bound by an immunoadhesin (Fc fusion protein) present in the subject. In some embodiments, the ligand moiety is bound by a circulating antibody. Circulating antibodies may be present in a subject due to naturally acquired immunity. Alternatively, circulating antibodies are present as a result of prior immunization of the subject. For example, circulating antibodies may be present as a result of childhood vaccination against smallpox, measles, rubella, herpes, hepatitis and polio. Thus, the ligand moiety may comprise one or more epitopes recognized by these circulating antibodies.

However, in some embodiments, the ligand moiety interacts with an antibody that has been administered to the subject. For example, an antibody that interacts with the ligand portion of an adapter molecule of the present invention can be co-administered with the adapter molecule. In addition, antibodies that interact with the ligand moiety may not normally be present in a subject, but the subject has obtained the antibodies by introducing biological material or antigen (e.g., serum, blood, or tissue) to produce high titers of antibodies in the subject. For example, a subject undergoing blood transfusion obtains a plurality of antibodies, some of which can interact with the ligand portion of the adapter peptide.

The ligand moiety may comprise any compound capable of binding an antibody, including but not limited to a peptide, carbohydrate, lipid, antibody or antibody-like molecule. In some embodiments, a ligand moiety (e.g., a peptide, antibody, or antibody-like molecule) can comprise one or more unnatural amino acids. Preferably, the ligand moiety comprises an epitope that binds "high titer antibodies". The term "high titer epitope" as used herein refers to an antibody having a high affinity for an antigen (e.g., an epitope on an antigenic domain). For example, in a solid phase enzyme-linked immunosorbent assay (ELISA), high titers of antibodies correspond to antibodies present in a serum sample that remain positive in the assay after the serum is diluted to a range of about 1: 100 to 1: 1000 in an appropriate dilution buffer. Other dilution ranges include 1: 200-1: 1000, 1: 200-1: 900, 1: 300-1: 800, 1: 400-1: 700, 1: 400-1: 600, and the like. In certain embodiments, the ratio between serum and dilution buffer is about: 1: 100, 1: 150, 1: 200, 1: 250, 1: 300, 1: 350, 1: 400, 1: 450, 1: 500, 1: 550, 1: 600, 1: 650, 1: 700, 1: 750, 1: 800, 1: 850, 1: 900, 1: 950, 1: 1000.

In certain embodiments, the ligand moiety is an antigenic peptide obtained from a known target molecule of an antibody (e.g., a surface protein from a pathogen, tumor cell, or infected host cell). The length of the peptide ligand moiety is desirably between at least 3-200 amino acids, preferably between 3-100 amino acids, more preferably between 3-50 amino acids, still more preferably between 10-25 amino acids (e.g., 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids). In some embodiments, the peptide consists of natural amino acids. In other embodiments, the peptide includes one or more unnatural amino acids (e.g., D-amino acids).

In certain embodiments, the ligand moiety is a glycosylated ligand moiety. The glycosylated ligand moiety may comprise or consist of an antigenic saccharide or glycan moiety recognized by an antibody in the subject. In some embodiments, the glycosylated ligand moiety is linked directly or indirectly (i.e., through a linker moiety) to the targeting moiety of the adapter molecule. This ligand moiety lacks an antigenic peptide moiety. In other embodiments, the glycosylated ligand moiety is a glycopeptide that comprises an additional antigenic peptide element (e.g., an antigenic domain comprising a peptide or epitope of a pathogen).

Exemplary glycosylated ligand moieties are derived from blood group antigens. These antigens are usually surface markers located outside the cell membrane of erythrocytes. Most of these surface markers are proteins, however, some are carbohydrates bound to lipids or proteins. Structurally, the blood group determinants that can be used in the embodiments described herein fall into two basic categories, namely type I and type II. Type I comprises a backbone consisting of galactose 1-3 β linked to N-acetylglucosamine, while type II comprises 1-4 β linkages between the same building blocks. The location and extent of fucosylation of these backbone structures gives rise to Lewis-type (Lewis-type) and H-type specificity. For example, the presence of a-monofucosyl branches only at the C2-hydroxyl group in the galactose moiety in the backbone constitutes H-type specificity (types I and II), while further alignment by substitution of a-linked galactose or a-linked N-acetylgalactosamine provides the molecular basis for the well-known serological blood group classifications A, B and O. By first determining the patient specific blood group antigen set one can select a ligand part comprising one or more blood group antigens other than the whole type of the patient to generate a strong reaction to an adapter molecule comprising this ligand part and thus redirect the antibodies present in the patient to the target molecule bound by the targeting part of the adapter molecule. Exemplary blood group antigens are described in detail in table 2 of U.S. patent No. 7,318,926, which is incorporated herein by reference in its entirety.

In certain preferred embodiments, the glycosylated ligand moiety comprises one or more gal- α -1-3gal disaccharide sugar units of a gal antigen. gal antigens are produced in large quantities on the cell surface of pigs, mice and new world monkeys by the glycosylase galactosyltransferase (α (1, 3) GT). Since humans and old world primates lack Gal antigens, they are not immune tolerant to Gal antigens and produce anti-Gal antigen antibodies (anti-Gal) throughout life in response to antigenic stimulation by gastrointestinal bacteria. Anti-gal antibodies have been estimated to represent more than 2% of circulating IgG and 1-8% of circulating IgM in humans. The binding of anti-Gal to Gal antigens expressed on glycolipids and glycoproteins on the endothelial cell surface of donor organs leads to activation of the complement cascade and hyperacute rejection and plays an important role in the development of complement-dependent late-onset xenograft rejection. Therefore, gal antigens have the ability to generate potent immune responses.

In certain preferred embodiments, the glycosylated ligand moiety to be bound or incorporated into the adaptor molecule consists essentially of one or more Gal-a (1-3) -Gal disaccharide sugar units and lacks any remaining portion of the Gal antigen (e.g., GlcNac or Glc). For example, one or more Gal- α (1-3) -Gal disaccharides can be linked to the targeting moiety of the adapter molecule through a free hydroxyl group at the reducing end of the disaccharide unit (e.g., a hydroxyl group at the C1 carbon that is not involved in a glycosidic bond). In certain embodiments, the Gal disaccharide is linked to the targeting moiety at the free hydroxyl group by a spacer moiety (e.g., a C1-C6 alkyl spacer). Without being bound to any particular theory, it is believed that the Gal- α (1-3) -Gal disaccharide moiety of the Gal antigen is preferentially linked to the anti-Gal antibody, but shows limited binding to lectins (e.g., Galectin-3) or other molecules that bind to other moieties of the Gal antigen (e.g., Gal- β (1-4) -GlcNAc moieties). In other embodiments, the glycosylated ligand moiety to be bound or incorporated into the adaptor molecule comprises an additional sugar residue of the Gal antigen. For example, one or more trisaccharide (Gal- α (1-3) -Gal- β (1-4) -GlcNAc or Gal- α (1-3) -Gal- β (1-4) -Glc), tetrasaccharide (Gal- α (1-3) -Gal- β (1-4) -GlcNAc- β (1-3) -Gal), or pentasaccharide (Gal- α (1-3) -Gal- β (1-4) -GlcNAc- β (1-3) -Gal- β (1-4) -Glc) units of the Gal antigen can be incorporated.

In certain embodiments, the Gal antigen to be bound or incorporated into the adaptor molecule is selected from the group consisting of a novel glycoprotein of the Gal-a- (1, 3) Gal series and may include Gal α 1-3Gal-BSA (3-atom spacer), Gal α 1-3Gal-BSA (14-atom spacer), Gal α 1-3Gal-HSA (3-atom spacer), Gal α 1-3Gal-HSA (14-atom spacer), Gal α 1-3Gal β 1-4GlcNAc-BSA (3-atom spacer), Gal α 1-3Gal β 1-4GlcNAc-BSA (14-atom spacer), Gal α 1-3Gal β 1-4GlcNAc-HSA (3-atom spacer), Gal α 1-3Gal β 1-4GlcNAc-HSA (14-atom spacer), Gal α 1-3 Gal-pentasaccharide-BSA (3-atom spacer), etc. In other embodiments, the Gal antigen may be selected from Gal α (1, 3) Gal analog neoglycoproteins including Gal α 1-3Gal β 1-4Glc-BSA (3-atom spacer), Gal α 1-3Gal β 1-4Glc-HSA (3-atom spacer), Gal 1-3Gal β 1-3GlcNAc-BSA (3-atom spacer), Gal α 1-3Gal β 1-3GlcNAc-HSA (3-atom spacer), Gal α 1-3Gal β 1-4 (3-deoxyglcnac) -HSA (3-atom spacer), Gal α 1-3Gal β 1-4 (6-deoxyglcnac) -HAS, and the like.

In still other embodiments, a peptide mimetic of a Gal antigen can be incorporated into an adapter molecule of the invention. Exemplary peptidomimetics include the α Gal-linked glycopeptides Gal- α -YWRY, Gal- α -TWRY, and Gal- α -RWRRY. Other peptidomimetics can be identified by screening randomized libraries of α Gal glycopeptides for anti-Gal antibody binding activity using the method of Xiaoan et al (see J. Comb. chem., 6: 126-134(2004), incorporated herein by reference).

The Gal antigen, or a peptidomimetic thereof, can be linked (covalently and/or non-covalently) to the targeting moiety using any art-recognized means. Art-recognized non-covalent linkages include biotin-avidin or biotin-streptavidin linkages and other high affinity binding partners (e.g., leucine zippers, etc.). Suitable means of covalent attachment include, but are not limited to, those described in figure 4 and in U.S. patent application No. 20100183635, which is incorporated herein by reference in its entirety. For example, the gal antigen (or peptidomimetic) can be directly linked to the polypeptide backbone of the polypeptide targeting moiety through a synthetic chemical linker. Exemplary synthetic chemical linkers include bifunctional linker moieties, such as linkers having maleimide functionality. For example, the bifunctional linker moiety may link the targeting moiety and the ligand moiety via an amino group in the targeting moiety and a thiol moiety in the ligand moiety, or vice versa. In an exemplary embodiment, a maleimide linker (e.g., thio-SMCC) is used to attach the cysteine residue of the polypeptide targeting moiety to the amino-modified Gal antigen (e.g., B of fig. 5)_di-(CH₂)₃-NH₂) On the amino group of (a). Additionally or alternatively, the gal antigen may be linked to an N-linked oligosaccharide of the glycoprotein targeting moiety. In some embodiments, one or more Gal antigens (e.g., Gal- α - (1, 3) Gal) or a peptidomimetic thereof are attached to a single site on the target moiety. In other embodiments, oneOne or more Gal antigens (e.g., Gal- α - (1, 3) Gal) or peptidomimetics thereof are attached to multiple sites on the target moiety. In certain embodiments, the linker moiety is chemically modified to reduce enzymatic or chemical degradation.

An adaptor molecule comprising a Gal antigen as disclosed herein is particularly advantageous because it does not significantly activate basophils and therefore does not cause IgE-mediated hypersensitivity when administered to a subject. Nevertheless, in some embodiments, the ability of the adaptor molecules of the invention to activate basophils is determined. Suitable assays for measuring basophil activation are known in the art (see, e.g., J.allergy Clin Immunol (2002)110102-9, which is incorporated herein by reference in its entirety).

In certain embodiments, the ligand moiety comprises a polymeric binding molecule, wherein the monomer is not an amino acid.

In certain exemplary embodiments, the high affinity adaptor molecule is selected from the group consisting of:

(a)H-Gly-D-Val-D-Gln-D-Glu-D-Asp-D-Val-D-Ser-D-Ser-D-Thr-D-Leu-Gly-D-Ser-D-Trp-D-Val-D-Leu-D-Leu-D-Pro-D-Phe-D-His-D-Arg-Gly-D-Thr-D-Arg-D-Leu-D-Ser-D-Val-D-Trp-D-Val-D-Thr-PEG₂-Cys-X-Y；

(b)H-Gly-Gly-D-Phe-D-Glu-Gly-D-Leu-D-Ser-D-Gln-D-Ala-D-Arg-D-Lys-D-Asp-D-Gln-D-Leu-D-Trp-D-Leu-D-Phe-D-Leu-D-Met-D-Gln-D-His-D-Ile-D-Arg-D-Ser-D-Tyr-D-Arg-D-Thr-D-Ile-D-Thr-PEG₂-Cys-X-Y；

(c) H-Gly-D-Val-Gly-Gly-Gly-D-Ser-D-Arg-D-Leu-D-Glu-D-Ala-D-Tyr-D-Lys-D-Lys-D-Asp-D-His-D-Arg-D-Val-D-Phe-D-Gln-D-Met-D-Ala-D-Trp-D-Leu-D-Gln-D-Tyr-D-Tyr-D-Trp-D-Ser-D-Thr-D-Thr-PEG 2-Cys-X-Y; and

(d)H-Gly-D-Ser-Gly-D-Ser-Gly-D-Asn-D-Ala-D-Leu-D-His-D-Trp-D-Val-D-Cys-D-Ala-D-Ser-D-Asn-D-Ile-D-Cys-D-Trp-D-Arg-D-Thr-D-Pro-D-Trp-D-Ala-Gly-D-Gln-D-Leu-D-Trp-Gly-D-Leu-D-Val-D-Arg-D-Leu-D-Thr-PEG2-Cys-X-Y；

wherein X is a bifunctional chemical linker having maleimide functionality; and Y is an amino-modified Gal-1-3-Gal disaccharide.

(c) Modified adaptor molecules

One or more portions of the adapter molecules of the present invention may be modified. In certain embodiments, the peptide portion of the adapter molecule is modified. For example, the peptide portion of the adapter molecule may be modified to include unnatural amino acids, such as those described in U.S. patent No. 6,559,126, which is incorporated herein by reference. For example, the peptides of the invention may be composed of one or more, or most preferably all, amino acids that are D-type optical isomers. These D-peptides have several benefits over antibodies and other protein therapeutics. The smaller size and higher stability of the D-peptide makes it easier to formulate for pulmonary, topical, and oral delivery. D-peptides are also known as weak immunogens (Dintzis et al, (1993) PROTECTINS: Structure, Function, and Genetics 16, 306-308). Furthermore, the resistance of D-peptides to enzymatic degradation and their ability to bind to polymers leads to enhanced pharmacokinetics compared to other peptide drugs. Furthermore, D-peptide has a reduced cost of growth, which advantage can be transmitted to the consumer.

The peptide component of the adaptor molecule may also be modified by a variety of standard chemical methods (e.g., amino acids may be modified with protecting groups; the carboxy-terminal amino acid may be formed as a terminal amide group; the amino-terminal residue may be modified with groups, e.g., to enhance lipophilicity; or the polypeptide may be chemically glycosylated or otherwise modified to increase stability or in vivo half-life). Adapter molecules of the invention can be designed to include chemical modifications or specific amino acid sequences that promote solubility. For example, in some embodiments, the peptide portion may be synthesized to include the amino acids DDD or KKK at the N-terminal or C-terminal region. Additionally or alternatively, the peptide or other targeting moiety may be synthesized to include a PEGylated moiety, for example at the N-terminal and/or C-terminal regions. Exemplary PEGylated moieties include PEG₂-NH₂And PEG₂-Cys-NH₂And (4) partial.

The invention also encompasses "conservative sequence modifications" or "conservative amino acid modifications" of the sequences described herein, i.e., amino acid sequence modifications that do not significantly affect or alter the binding properties of a peptide encoded by or comprising the nucleotide sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can also be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. In some embodiments, the modifications are selected by rational design, and the designed peptides are produced by chemical synthesis as described herein. "conservative amino acid modifications" include conservative amino acid substitutions as substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain (e.g., similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

The peptides of the invention or mimetics thereof may be modified by one or more substitutions, particularly in the portion of the protein that is not expected to interact with the protein of interest. It is contemplated that up to 5%, 10%, 20%, 30%, 40%, 50% or even more than 50% of the amino acids in a peptide can be altered by conservative substitutions without substantially altering the affinity of the protein for the target protein. It is possible that such changes alter the in vivo immunogenicity of the polypeptide, and in cases of reduced immunogenicity, such changes are desirable. Additional non-limiting examples of analogous substitutions that can be made in the molecular structure of the peptides of the invention include the substitution of D-phenylalanine with D-tyrosine, D-pyridylalanine, or D-homophenylalanine, the substitution of D-leucine with D-valine or other natural or unnatural amino acid having an aliphatic side chain, and/or the substitution of D-valine with D-leucine or other natural or unnatural amino acid having an aliphatic side chain. In some embodiments, conservative amino acid substitutions alone, i.e., without amino acid deletions or additions, are a preferred type of amino acid modification. Those skilled in the art will appreciate that such modifications or substitutions may be made at the DNA level, thus encoding altered or substituted peptides, or they may be made at the protein level, for example by direct chemical synthesis.

In some embodiments, the peptide or peptide portion of the adapter molecule can be made cyclic. Such "cyclic peptides" have an intramolecular linkage connecting two amino acids. Cyclic peptides are often resistant to proteolytic degradation and are therefore good candidates for oral administration. The intramolecular linkage may comprise an intermediate linking group or may involve direct covalent bonding between amino acid residues. In some embodiments, the N-terminal and C-terminal amino acids are linked. In other embodiments, one or more internal amino acids participate in the cyclization. Other methods known in the art may be used to cyclize the peptides of the invention. For example, cyclic peptides can be formed by the side chain azide-alkyne 1, 3-dipolar cycloaddition reaction (Cantel et al, J.org.chem., 73(15), 5663-5674, 2008, incorporated herein by reference). For example, the compositions may also be prepared by the methods of U.S. Pat. Nos. 5596078; 4033940, respectively; 4216141, respectively; 4271068, respectively; 5726287, respectively; 5922680, respectively; 5990273, respectively; 6242565, respectively; and Scott et al, PNAS.1999.vol.96 No.24 p 13638-13643. In some embodiments, the intramolecular link may be a disulfide bond mimetic or a disulfide bond mimetic that retains a structure otherwise created by a disulfide bond.

In some particularly preferred embodiments, cyclization of the peptide occurs through an intramolecular disulfide bond. In some preferred embodiments, intramolecular disulfide bond formation increases the affinity of the peptide. Thus, the methods for selecting and/or affinity-maturing a peptide of the present invention or a mimetic thereof may be performed under conditions that allow disulfide bond formation (e.g., oxidizing conditions) prior to or during selection. In some particularly preferred embodiments, disulfide bonds may be formed between cysteine residues naturally occurring in the library or peptide, or introduced by mutagenesis methods during one or more rounds of selection. In other embodiments, the peptides may be designed to contain cysteine residues at specific positions so that it is possible to know which residues are involved in disulfide bonds. Intramolecular disulfide bonding between cysteine residues can be induced by methods known in the art (e.g., U.S. Pat. Nos. 4572798; 6083715; 6027888, and WIPO publication WO/2002/103024, incorporated herein by reference).

In some embodiments, the formation of disulfide bonds (or formation of structures that are generally cyclized or intramolecular linked) confer specific structures on the peptide that are important for target binding. Thus, it is preferred that disulfide bonds and/or cyclization are formed prior to peptide selection, so that potentially advantageous structures resulting from bond formation can be selected. In some embodiments, a peptide of the invention or a mimetic thereof may have more than one, two, three, or more disulfide bonds. Other methods known in the art for generating and selecting peptides having intramolecular disulfide bonds, intramolecular disulfide bond substitutes, or other intramolecular linkages can be used. For example, the methods described in WO03040168, incorporated herein by reference, describe methods of producing and selecting peptide aptamers, conjugates, and other cyclized peptides, which in some embodiments can be used in the methods of the invention.

In related embodiments, peptide conformations or structures that are beneficial for binding (e.g., increased binding affinity) can be maintained or mimicked by chemical crosslinking or other methods of peptide stabilization. For example, a beneficial peptide conformation or structure formed by disulfide bonds may be stabilized by chemical treatment or reaction such that the structure is maintained without disulfide bonds. Indeed, peptide stabilization techniques may be used to stabilize the peptides of the invention, whether or not disulfide bonds are originally present. For example, Jackson et al, J.Am.chem.Soc.1991, 113, 9391-9392; phelan et al, J.Am.chem.Soc.1997, 119, 455-460; the technique described in Bracken et al, J.Am.chem.Soc.1994, 116, 6431-.

Other methods of stabilizing peptides and peptide structures may be used, for example, olefinic crosslinking through helices of o-allylserine residues (Blackwell, H.E.; Grubbs, R.H.Angew.chem., int.Ed.1998, 37, 3281-3284, incorporated herein by reference), all hydrocarbon crosslinking (Schafmeister and Verdine J.am.chem.Soc.2000, 122(24), 5891-5892, incorporated herein by reference), and the methods disclosed in U.S. Pat. No. 7183059 (incorporated herein by reference). The methods disclosed in Blackwell et al and Schaffeister et al can be described as producing "stapled" peptides, i.e., peptides that are covalently locked in a particular conformational state or secondary structure, or peptides that are covalently linked intramolecularly that have a tendency to form a particular conformation or structure. If the peptides so treated are prone to, for example, formation of an alpha-helix important for target binding, the energy threshold for binding will be reduced. Such "stapled" peptides have been shown to be resistant to proteases and may also be designed to more efficiently cross cell membranes (see also Walensky et al, Science 2004: Vol.305.no.5689, p. 1466-1470; Bernal et al, J Am Chem Soc.2007, 129 (9): 2456-7, incorporated herein by reference). Thus, the peptides or peptide portions of the invention may be stapled or otherwise modified to lock them in a particular conformational shape or they may be modified to a particular conformation or secondary structure that tends to favor binding. It is contemplated that such peptide modifications may occur prior to peptide selection, so that any conformational constraint benefit may also be selected. Alternatively, in some embodiments, modifications may be made after selection to retain a conformation known to be beneficial for binding or to further enhance the peptide candidate.

In other embodiments, the ligand portion of the adapter molecule may be modified. The modification may be carried out, for example, by dryingThe interfering molecule minimizes competitive binding or reduces enzymatic or chemical degradation of the ligand moiety (e.g., under physiological conditions). As used herein, the term "interfering molecule" refers to a binding molecule (e.g., circulating or cell surface receptor) that competes with circulating antibody for binding to the adaptor molecule and prevents its intended therapeutic effect from being exerted (e.g., by rapid clearance of the adaptor molecule from the circulation). For example, the glycosylated ligand may comprise a Gal antigen or mimetic that has been chemically modified to enhance preferential binding by anti-Gal antibodies while minimizing undesired binding by lectins (e.g., galectin-3) or other interfering molecules. Additionally or alternatively, the glycosylated ligand moiety may comprise a gal antigen or mimetic that has been chemically modified, for example to reduce enzymatic or chemical degradation or to facilitate covalent attachment. Exemplary modifications include the addition of biologically inert protecting groups to the reactive hydroxyl groups on the sugar residues of the gal antigen, for example, by anhydrocoupling, reductive amination, or enzymatic oxidation, for example, with galactose oxidase. A protective group can be added to the C-6' OH of the terminal Gal residue of the Gal epitope (see, e.g., Andreana et al, glycon project J., 20: 107-118 (2004)). Exemplary protecting groups include amine (e.g., aminopyridine) and oxime substituents (e.g., O-Me-oxime, O-Et-oxime, O-tBu-oxime, O-Bn-oxime, and O-allyl-oxime). Alternatively, the polar C-6' OH group may be replaced by a non-polar hydrogen to form a 6-deoxy- α -Gal derivative (see Janczuk et al, Carbohydrate Research, 337: 1247-1259(2002), incorporated herein by reference). In certain exemplary embodiments, the Gal epitope can be modified with an amino group (e.g., alkyl-NH)₂Substituent) to facilitate attachment to the targeting moiety (e.g., at C-1 OH). The binding of the anti-Gal antibody to the modified Gal antigen can then be assessed using art-recognized methods (e.g., ELISA).

(d) Multivalent linker molecules

It is contemplated that multiple peptides or peptide moieties of the species disclosed herein can be ligated to create a composite adaptor molecule with increased affinity or binding valency. Likewise, the peptide or peptide portion of the adapter molecule can be linked to a variety of other polypeptides, such as fluorescent polypeptides, targeting polypeptides, and polypeptides with distinctly different therapeutic effects.

Methods for identifying high affinity adaptor molecules

In certain aspects, the invention provides methods for identifying adaptor molecules with high binding affinity or selectivity. The methods of the invention comprise (i) at least one selection step to identify high affinity targeting moieties (e.g., targeting peptide moieties and/or ligand peptide moieties), and (ii) a ligation step, wherein the targeting moiety and ligand moiety of an adaptor molecule are ligated to form an adaptor molecule.

In certain embodiments, the methods of the invention utilize ribosome display or mRNA display as a selection step to identify one or more targeting moieties of an adaptor molecule. A general overview of ribosome display and mRNA display Methods is provided by Lipovsek and Pluckthun (J.immunological Methods, 290: 51-67(2004)) which are incorporated herein by reference in their entirety. In preferred embodiments, mRNA display is used to identify targeting moieties (e.g., peptide targeting moieties) of adapter molecules. An exemplary mRNA display method is depicted in fig. 2. Briefly, the starting library is obtained, for example, by direct DNA synthesis or by in vitro or in vivo mutagenesis. The double stranded DNA library is then transcribed in vitro (e.g., using T7 polymerase) and ligated to a puromycin-like linker. In vitro translation is performed, wherein the puromycin-like linker is reacted with the nascent translation product. After purification, a high degree of diversity (. about.10) was obtained¹³) A library of peptide-RNA fusion molecules of (1). Reverse transcription produces a cDNA/RNA hybrid covalently linked to the transcribed peptide. This complex is then selected by using the molecule of interest (in the case of a targeting peptide moiety) or an antibody (in the case of a ligand peptide moiety). The peptides that bind the target or antibody molecule are selected (e.g., under stringent washing conditions), and the cDNA is conveniently eluted to identify the selected peptides. The selection can be performed multiple times to identify high affinity binders. It should be noted that in the selection process, the selection process may be performed under conditions such that intramolecular disulfide bonds are present in the peptide. In other embodiments, ifIf desired, the formation of disulfide bonds can be prevented.

In additional or alternative embodiments, the methods of the invention utilize phage display and/or yeast display technology as a selection step to identify one or more targeting (e.g., peptide) portions of an adapter molecule. Non-limiting examples of such library screening methods are described in, for example, U.S. Pat. nos. 7,195,880; 6,951,725, respectively; 7,078,197, respectively; 7,022,479, respectively; 5,922,545, respectively; 5,830,721; 5,605,793; 5,830,650, respectively; 6,194,550; 6,699,658.

In other embodiments, the methods of the invention utilize a library of synthetic peptides. Such synthetic peptides can be chemically synthesized or enzymatically produced (e.g., by in vitro translation of RNA or by enzymatic digestion of a preexisting protein). In some embodiments, the library of synthetic peptides is arranged on a solid phase substrate (e.g., a glass slide).

In certain embodiments, the methods of the invention utilize mRNA libraries (e.g., mRNA display libraries) encoding randomized peptides corresponding to portions of larger polypeptides known to interact with a particular target or antibody molecule. An exemplary mRNA display library is depicted in fig. 3. For example, a peptide library may comprise a population of linear peptide molecules in which the amino acid sequence of the peptide is randomized at one or more amino acid positions (preferably at least 10 or more amino acid positions) within the molecule. The randomized portion of such a peptide sequence may be flanked by one or more constant regions from the parent polypeptide.

In certain embodiments, the methods of the invention comprise providing an mRNA display library encoding a randomized population of candidate targeting moieties (e.g., or peptides or polypeptides). By way of example, the targeting peptide may be a randomized peptide derived from a soluble ligand, such as VEGF. High affinity targeting peptides can be selected by screening peptide-RNA-cDNA fusions from a library. Preferably, multiple selection cycles are performed to enrich the population of molecules that bind to the molecule of interest (e.g., VEGF receptor). By reducing the concentration of target molecules in the respective selection steps, peptides that bind with the highest affinity to the target molecules can be further enriched in the population. Additional selection processes that may be utilized in the various selection steps include: (1) counter-selection to eliminate non-specific peptides; (2) competitive elution to identify site-specific peptides; (3) and selection under specific solution conditions (e.g., high stringency wash conditions) to identify stable peptides.

In certain embodiments, library members are modified prior to selection to include a coupling moiety that can react with a linker (e.g., a bifunctional linker) to form a linking moiety. In other embodiments, the library members are modified with a coupling moiety after the selection step to facilitate attachment to the linking moiety. Exemplary coupling moieties include terminal amino acids (e.g., a C-or N-terminal cysteine or cysteine analog) or amino acid side chains (e.g., a cysteine or cysteine analog side chain) that can be reacted with a maleimide functional bifunctional linker.

Once the high affinity targeting moiety has been identified, the peptide can be linked to a preselected ligand moiety through a linking moiety, thereby generating an adaptor molecule. In certain embodiments, the ligand moieties have been pre-selected using mRNA display methods. Alternatively, the targeting moiety may be inserted as a constant region into a second mRNA display library encoding a randomized population of candidate ligands. This second mRNA display library can then be subjected to a further selection step in which the library members are screened against antibodies to identify adapter molecules. Thus, the candidate ligand preferably corresponds to a portion (e.g., an epitope) of an antibody ligand. In one embodiment, the antibody ligand moiety may be an epitope to which the antibody binds. In another embodiment, the antibody ligand moiety may be a unique site (idiotope) of the primary antibody to which the second anti-idiotype antibody binds. In yet another embodiment, the antibody ligand portion can be an Fc binding portion of an Fc binding protein (e.g., an Fc receptor).

After selecting an adapter molecule that binds the target molecule and the antibody molecule with high affinity and/or selectivity, the ability of the adapter molecule to redirect antibody specificity to the target molecule can be evaluated. For example, where the target molecule is a cell surface molecule, the ability of the adaptor molecule to induce effector function (e.g., antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)) and cell killing can be assessed using art-recognized techniques.

In still other embodiments, library members are linked to a ligand moiety prior to the selection step. For example, each member of the library to be screened may be derivatized with a Gal antigen, followed by a screening step to identify high affinity adaptor molecules. The Gal antigen may be linked to the terminal amino acid or amino acid side chain of each peptide.

In still other embodiments, an iterative selection method may be utilized in which a targeting moiety is selected in a first selection step (or in a first series of selection steps) and the sequence of the targeting moiety is incorporated into the constant region of the mRNA-peptide fusion to facilitate selection of the ligand moiety in a second selection step (or in a second series of selection steps). For example, alternating first and second selection steps (or series of selection steps) in successive rounds of selection allows for the identification of high affinity adaptor molecules.

Methods for synthesizing high affinity adaptor molecules

Once the components of the high affinity adaptor molecules have been identified using the methods described herein, they can be prepared using standard methods known in the art. For example, the peptide can be prepared by recombinant DNA methods, inserting a nucleic acid sequence (e.g., cDNA) encoding the polypeptide into a recombinant expression vector and expressing the DNA sequence under conditions that promote expression. General techniques for nucleic acid manipulation are described, for example, in Sambrook et al, Molecular Cloning: a Laboratory Manual, Vols.1-3, Cold Spring Harbor Laboratory Press, 2ed., 1989, or F.Ausubel et al, Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and periodic updates. Suitable Cloning and expression Vectors for bacterial, fungal, yeast and mammalian cell hosts may be Cloning Vectors: a Laboratory Manual, (Elsevier, New York, 1985), the relevant disclosure of which is incorporated herein by reference. Other recombinant DNA methods are described in U.S. patent nos. 4356270, 4399216, 4506013, 4503142, 4952682, 5618676, 5854018, 5856123, 5919651, and 6455275, which are incorporated herein by reference.

Adapter molecules and components thereof can also be prepared by chemical synthesis using techniques well known in the art. For example, D-peptides can be synthesized using stepwise addition of D-amino acids in a solid phase synthesis process involving the use of suitable protecting groups. Solid phase peptide synthesis techniques commonly used for L-Peptides are incorporated herein by reference in their entirety, Meinhofer, Horminonal Proteins and Peptides, vol.2, (New York 1983); kent et al, ann.rev.biochem., 57: 957 (1988); bodanszky et al, Peptide Synthesis, (2d ed. 1976); atherton et al, (1989) Oxford, England: IRL press. isbn 0199630674; stewart et al, (1984) second edition, Rockford: pierce Chemical Company, 91.ISBN 0935940030 and Merrifield (1963) j.am.chem.soc.85: 2149, 2154. D-amino acids for use in solid phase synthesis of D-peptides are available from a variety of commercial sources. D-peptides and peptides comprising mixed L-and D-amino acids are known in the art. Also, peptides containing exclusively D-amino acids (D-peptides) have been synthesized. See Zawadzke et al, j.am.chem.soc., 114: 4002-4003 (1992); milton et al, Science 256: 1445-1448(1992). Other methods for preparing D-peptides have been described in the art and may be found in at least WIPO publication No. WO/1997/013522 and U.S. application No. 60/005,508, which are incorporated herein by reference.

The peptide of the present invention can be purified by a protein isolation/purification method generally known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination thereof. After purification, the peptide may be exchanged into a different buffer and/or concentrated by any of a variety of methods known in the art, including, but not limited to, filtration and dialysis. The purified polypeptide is preferably at least 85% or 90% pure, more preferably at least 93% or 95% pure, and most preferably at least 97%, 98%, or 99% pure. Regardless of the exact numerical value of purity, the peptide is sufficiently pure to be useful as a pharmaceutical product.

Examples

The disclosure is further illustrated by the figures and the following examples (which should not be construed as further limiting). The contents of all figures and all references, patents and published patent applications cited in this application are hereby incorporated by reference in their entirety.

Example 1 selection of high affinity anti-VEGF peptides as targeting moieties and binding to alpha Gal ligand moieties

The library of randomized peptide sequences was screened for high affinity binding to VEGF using the mRNA display method described in figure 2. Four high affinity anti-VEGF peptide sequences (SEQ ID NOS 1-4) were selected. Each peptide was C-terminally attached with PEG₂-NH₂Or PEG₂-Cys-NH₂Partial PEGylation (see FIG. 4) to facilitate conjugation to Gal antigen (Bdi- (CH)₂)-NH₂Disaccharide).

To facilitate chemical binding of each peptide to Gal disaccharide, a bifunctional linker with maleimide and thio-NHS functionalities (thio-SMCC, PIERCE) was employed. The linker reacts with the amino groups present in the disaccharide and the maleimide functionality in the thiol (thiol) group of the C-terminal cysteine residue of the peptide. To obtain the desired compound and avoid any further reactions and by-product formation, the reaction was RP-HPLC purified immediately after incubation. The ratio of the amounts of the reactants is optimized to avoid the formation of by-products. In an exemplary synthesis, 6mg of disaccharide compound B_di-(CH₂)-NH₂(. about.15. mu. mol) was dissolved in 100. mu.l of 0.1M Hepes buffer containing 20% MeCN, pH 6.0; 1.3mg of thio-SMCC linker (. about.3. mu. mol) was dissolved in 100. mu.l of 0.1M H containing 20% MeCNepes buffer, pH 6.0; and the two solutions were mixed and incubated at room temperature for 30 minutes with continuous rotation of the reaction tube. 1mg of peptide 07-090 (07-090; lyophilized powder, TFA adduct, M.W.3474g/mol; -320 nmol) was dissolved in 1ml of H₂O/MeCN 80: 20%. The clarified peptide solution was added to the linker-disaccharide solution immediately after preparation and incubated for an additional 90 minutes at room temperature with continuous rotation of the reaction tube. The reaction mixture was placed on ice and an aliquot was analyzed on an RP-HPLC-C18 column. The content of the active carbon is 0-50%; and (3) buffer solution A: h₂O/5% MeCN, 0.1% TFA, buffer B: h₂HPLC gradient of O/5% MeCN, 0.1% TFA run on the expected compound (B)_di-(CH₂) -NH-linker-S-07-090-peptide). HPLC fractions were analyzed by ESI-TOF-MS after dilution with 65% methanol, 0.5% formic acid, and product fractions were identified. The relevant product peak fractions were frozen at-80 ℃ and then lyophilized to complete dryness to contain the desired compound.

Example 2 anti-VEGF-specific adaptor molecules can relocate native anti- α Gal antibodies to VEGF

Analytical methods were designed to test the ability of α Gal-linked anti-VEGF peptides to redirect native antibodies specific for α Gal to bind VEGF. The assay was designed with recombinant VEGF on a solid phase. Serial dilutions of α Gal-linked anti-VEGF peptides were then incubated with rVEGF, followed by high levels of anti- α Gal-containing serum from mice. The amount of bound anti- α Gal is then indicated with an enzyme-linked anti-mouse antibody. The presence of the enzyme is indicated by the addition of a chromogenic substrate and the increase in color is measured by measuring the optical density at 490 nm. The data shown in fig. 8 clearly demonstrate that α Gal-linked anti-VEGF peptides can redirect native anti- α Gal antibodies to VEGF due to a decrease in optical density resulting from a decrease in the amount of peptide or antisera.

Claims (33)

1. A method for identifying high affinity adaptor molecules capable of redirecting antibody specificity, the method comprising:

(a) providing a randomized library encoding a population of candidate targeting peptides;

(b) selecting from the display library targeting peptides that bind with high affinity and/or selectivity to the target molecule;

(c) linking the targeting peptide and the ligand moiety through a linking moiety to form a candidate adaptor molecule; and

(d) evaluating the ability of the candidate adaptor molecule to redirect circulating antibody specific to the target molecule;

thereby identifying the adaptor molecule.

2. The method of claim 1, wherein steps (a) - (d) are performed continuously.

3. The method of claim 1 or 2, wherein the linking step (c) is performed before step (b).

4. The method of any of the preceding claims, wherein the library is an mRNA display, ribosome display, yeast display, phage display or synthetic peptide library.

5. The method of any one of the preceding claims, wherein the targeting peptide binds to a target molecule with a binding affinity of 1nM or less.

6. The method of any preceding claim, wherein the ligand moiety comprises a glycan moiety.

7. The method of any one of the preceding claims, wherein the ligand moiety is a blood group antigen.

8. The method of any of the above claims, wherein the ligand moiety is a gal antigen or an epitope thereof.

9. The method of claim 8, wherein the ligand moiety consists of one or more gal- α -1-3-gal disaccharide units.

10. The method of claim 8, wherein the ligand moiety is a modified gal antigen with a modification that reduces competitive binding of interfering molecules.

11. The method of claim 8, wherein the ligand moiety is a modified gal antigen with a modification that reduces enzymatic or chemical degradation.

12. The method of claim 10 or 11, wherein the modified gal antigen comprises a protecting group at C6' of the terminal galactose residue.

13. The method of any of the above claims, wherein the ligand moiety is a peptidomimetic of a gal antigen.

14. The method of any one of the preceding claims, wherein the ligand moiety is a peptide ligand moiety.

15. The method of claim 14, wherein the peptide ligand moiety comprises an epitope that is selectively bound by an antigen binding site of a circulating antibody.

16. The method of claim 15, wherein the peptide ligand moiety comprises a unique position of an antibody, wherein the unique position is selectively bound by a circulating anti-idiotypic antibody.

17. The method of claim 16, wherein the peptide ligand moiety comprises a binding site portion of an Fc binding protein.

18. The method of any of the above claims, wherein the peptide ligand moiety is produced by (i) providing a randomized mRNA display library encoding a population of candidate peptide ligand moieties; and (ii) selecting from the display library of step (i) a peptide ligand moiety that binds with high affinity and/or selectivity to circulating antibody.

19. The method of claim 18, wherein, prior to selection step (ii), the candidate peptide ligand moiety is fused to a targeting peptide.

20. The method of claim 18, wherein, after selection step (ii), the candidate peptide ligand moiety is fused to a targeting peptide.

21. The method of any of the preceding claims, wherein the target molecule is a soluble disease-associated molecule.

22. The method of claim 21, wherein the redirected antibody specificity is assessed by measuring opsonization or neutralization of the soluble molecule.

23. The method of any of the preceding claims, wherein the ligand moiety is linked to the targeting moiety with a bifunctional linker moiety.

24. The method of claim 23, wherein the bifunctional linker moiety connects the targeting moiety and the ligand moiety through an amino group of the targeting moiety and a thiol moiety of the ligand moiety.

25. The method of any of the preceding claims, wherein the target molecule is present on the surface of an infected cell or a tumor cell.

26. The method of claim 25, wherein the redirected specificity is assessed by measuring ADCC or CDC-dependent cell killing.

27. A high affinity adaptor molecule identified according to the method of any one of the preceding claims, comprising (i) a targeting moiety that binds with high affinity or selectivity to a target molecule, (ii) a ligand moiety that specifically binds to a circulating antibody; and (iii) a linker moiety linking the targeting moiety and the ligand moiety, wherein the adapter molecule facilitates functional interaction between the antibody and the target molecule.

28. The high affinity adaptor molecule of claim 27, wherein the targeting moiety is a peptide targeting moiety.

29. The adaptor molecule of claim 27 or 28, wherein the targeting moiety binds to a VEGF ligand with high affinity or selectivity.

30. The adaptor molecule of any one of claims 27-29, wherein the targeting moiety comprises one or more sequences selected from SEQ ID NOs 1, 2, 3 and 4.

31. The adaptor molecule of any one of claims 27-29, wherein the targeting moiety is pegylated.

32. The adaptor molecule of any one of claims 27-30, wherein the ligand moiety comprises a Gal antigen that specifically binds to a circulating anti-Gal antibody.

33. A high affinity adaptor molecule selected from the group consisting of:

(a)H-Gly-D-Val-D-Gln-D-Glu-D-Asp-D-Val-D-Ser-D-Ser-D-Thr-D-Leu-Gly-D-Ser-D-Trp-D-Val-D-Leu-D-Leu-D-Pro-D-Phe-D-His-D-Arg-Gly-D-Thr-D-Arg-D-Leu-D-Ser-D-Val-D-Trp-D-Val-D-Thr-PEG₂-Cys-X-Y；

(b)H-Gly-Gly-D-Phe-D-Glu-Gly-D-Leu-D-Ser-D-Gln-D-Ala-D-Arg-D-Lys-D-Asp-D-Gln-D-Leu-D-Trp-D-Leu-D-Phe-D-Leu-D-Met-D-Gln-D-His-D-Ile-D-Arg-D-Ser-D-Tyr-D-Arg-D-Thr-D-Ile-D-Thr-PEG₂-Cys-X-Y；

(c) H-Gly-D-Val-Gly-Gly-Gly-D-Ser-D-Arg-D-Leu-D-Glu-D-Ala-D-Tyr-D-Lys-D-Lys-D-Asp-D-His-D-Arg-D-Val-D-Phe-D-Gln-D-Met-D-Ala-D-Trp-D-Leu-D-Gln-D-Tyr-D-Tyr-D-Trp-D-Ser-D-Thr-D-Thr-PEG 2-Cys-X-Y; and (D) H-Gly-D-Ser-Gly-D-Ser-Gly-D-Asn-D-Ala-D-Leu-D-His-D-Trp-D-Val-D-Cys-D-Ala-D-Ser-D-Asn-D-Ile-D-Cys-D-Trp-D-Arg-D-Thr-D-Pro-D-Trp-D-Ala-Gly-D-Gln-D-Leu-D-Trp-Gly-D-Leu-D-Val-D-Arg-D-Leu-D-Thr-2-Cys-X-Y;

wherein,

x is a bifunctional chemical linker having maleimide functionality; and

y is amino-modified Gal-1-3-Gal disaccharide.

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